The tissues on the surface of the eye and the secretory glands that are derived from these tissues are essential for vision. The lacrimal, Meibomian and conjunctival mucus glands secrete the components of the tear film. Insufficient function of any of these glands leads to dry eye disease. The corneal and limbal epithelia are responsible for maintaining the refractive surface of the eye. Deficiencies in the differentiation f the corneal epithelial cells or insufficient generation of corneal epithelial cells by the limbal sem cells leads to severe ocular irritation, inflammation, neovascularization of the corneal stroma and blindness. The aims of this proposal are 1) to identify the signaling systems that are responsible for the proper formation and function of the ocular surface epithelia and their derivatives during development and 2) to identify the signaling pathways that establish and maintain the limbal stem cells to provide functional corneal epithelial cells. The studies described in this proposal show that Pax6 and BMP signaling are key factors required for the formation and differentiation of all of the ocular surface epithelia. Laser microdissection and microarray analysis identified or confirmed four transcription factors that are early markers for the different ocular surface epithelia and are likely to play important roles in their differentiaton. Three of these factors depend on Pax6 for their expression. This proposal also describes a new method to genetically mark the limbal stem cells. At the same time, genes within these cells can be selectively deleted. This method will be used to inactivate critical pathways known to function in other adult stem cells. Three of these pathways, BMP and SDF-1 signaling and adhesion between niche and stem cells mediated by N-cadherin, have been shown by our collaborator on this project, Dr. Scheffer Tseng, to be involved in signaling between limbal stem and niche cells. The functions of these pathways will be tested in vivo by genetic ablation and confocal imaging. In each case, our vivo analyses will be complemented by in vitro genetic studies performed in Dr. Tseng's laboratory. This collaboration is possible because Dr. Tseng isolates limbal stem and adherent niche cells after overnight incubation. We will send eyes from our genetically-modified mice to Dr. Tseng by overnight courier. He will isolate the niche and stem cells and treat them with tamoxifen to delete the targeted genes. This collaboration will provide the most extensive analysis to date of the pathways that create and maintain the limbal stem cell niche. Information derived from both aims will be valuable for the replacement of injured or defective corneal and conjunctival epithelia using induced pluripotent cells or by reprogramming of other epithelial cell types.